The present invention relates to moleculy and newly identified polynucleotides and proteins as targets for blocking the activation of certain signal transduction pathways. Specifically, the invention provides methods and compositions for treating inflammatory disorders. More specifically, the invention provides methods for generating animals that have functionally disrupted Tpl2 gene, and methods and compositions targeted to the Tpl2 gene.
Protection from microbial pathogens is mediated by a variety of inducible effector mechanisms that are triggered upon encounter with pathogens. The sum of these mechanisms defines antimicrobial immunity which is subclassified into innate and adaptive (See Janeway, Jr. 1989, Cold Spring Harb. Symp. Quant. Biol. 54 Pt 1, 1-13). Innate immunity is triggered by pathogen-associated molecular patterns (PAMPs) which are shared by groups of microbial pathogens and which are recognized by pattern recognition receptors (PRRs) in host cells. (See Medzhitov et al., 1997a, Cell 91, 295-298). PRRs in turn include signaling and endocytic receptors, as well as secreted proteins which bind the microbes and facilitate their phagocytosis or destruction by the complement system. Pathogen-associated molecular patterns are represented by molecules localized on microbial walls. Prominent among them is lipopolysaccharide (LPS) or endotoxin which is derived from the Gram-negative bacteria. LPS consists of four distinct regions: the O-specific chain, the outer core, the inner core, and the lipid A moiety which is responsible for most of the biological effects attributed to LPS. (See Rietschel et al., 1992, Sci. Am. 267, 54-61). Other PAMPs include peptidoglycans which are present primarily in the walls of Gram-positive microorganisms, trehalose diesters which are produced by mycobacteria and corynebacteria, and other Gram-positive bacterial products such as lipotechoic acid. (See Springer, 1990, Nature 346, 425-434; Warren et al., 1986, Annu. Rev. Immunol. 4, 369-388; Lederer, 1979, Springer Semin. Immunopathol. 2, 133-148; Wicken et al., 1980, Biochim. Biophys. Acta 604, 1-26). LPS binds the soluble LPS-binding protein (LBD) and the complex binds CD14, a monocyte/macrophage receptor molecule that is expressed in both soluble and membrane-associated forms. CD14 presents the LPS-LBD complex to the LPS receptor TLR4 (See Medzhitov et al., 1997, Nature 388, 394-397), a member of the Toll family of receptors. TLR4 is the signaling receptor and responds to LPS even in the absence of CD14, whose role is to form, in combination with TLR4, a high affinity receptor.
There are at least 10 members of the Toll receptor family in mammals. (See Rock et al., 1998, Proc. Natl. Acad. Sci. U. S. A. 95, 588-593). Expression of one of these, TLR2, in 293 cells rendered them responsive to LPS suggesting that it is the LPS receptor. More interesting, while TLR4 recognizes LPS, TLR2 recognizes molecules derived from yeast and Gram-positive bacteria. (See Takeuchi et al., 1999, Immunity. 11, 443-451) There are two types of mouse models for the septic shock syndrome. In the first type the syndrome is induced by the administration of a large dose of LPS and is characterized by the development of symptoms over a period of several days to a week (See Fink et al., 1990, J Surg. Res. 49, 186-196; Wise et al, 1980, Circ. Res. 46, 854-859). Mice deficient in ICE (See Li et al., 1995, Cell 80, 401-411) or caspase-11 (See Wang et al., 1998, Cell 92, 501-509), which are required for the processing of IL-1xcex2 (See Black et al., 1989, FEBS Lett. 247, 386-390), are resistant to high dose LPS-induced shock, suggesting that IL-1xcex2 is the main cytokine responsible for this syndrome. In the second type of model, the syndrome is induced by the administration of a low dose of LPS combined with the transcriptional inhibitor D-Galactosamine (See Galanos et al., 1979, Proc. Natl. Acad. Sci. U. S. A 76, 5939-5943). This gives rise to a rapidly developing syndrome that culminates in death within 6-10 hours. The induction of TNFxcex1 by LPS in macrophages, the main cellular target for LPS/D-Galactosamine induced shock, is mediated by both transcriptional and posttranscriptional mechanisms. Particularly important in the posttranscriptional regulation of TNFxcex1 is an AU-rich element (ARE) in the 3-untranslated region of the TNFxcex1 mRNA (See Han et al., 1990, J Exp. Med 171, 465-475; Han et al., 1990, Eur. Cytokine Netw. 1, 71-75), which regulates the stability of the message and the efficiency by which it is translated (See Kruys et al., 1990, Enzyme 44, 193-202; Kruys et al., 1994, Biochimie 76, 862-866). This element represses translation.
Signals originating in the LPS-triggered TLR4 receptor activate several signaling pathways in target cells such as B cells, which respond by proliferation, and macrophages, which respond by expressing a large number of cytokines and other molecules. Such molecules target the pathogens either directly or indirectly by functionally modifying other cells (See Nathan, 1987, J Clin Invest 79, 319-326). Although these biological responses protect the host against invading pathogens, however, they may also cause harm. Thus, massive stimulation of innate immunity, occurring as a result of severe Gram-negative bacterial infections, leads to excess production of cytokines and other molecules, and the development of a fatal syndrome, the septic shock syndrome, which is characterized by fever, hypotension, disseminated intravascular coagulation and multiple organ failure. (See Parillo, 1993, N Engl J Med 328, 1471-1477). The present invention provides methods and compositions for treating the septic shock syndrome and other inflammatory diseases.
In the present invention, it has been found that biological and/or pharmacological agent induced and TNFxcex1-dependent septic shock syndrome (or endotoxin shock) and other inflmmatory diseases in animals including humans can be treated by eliminating or interfering with the functional role of the endogenous Tpl2 gene. In general, the present invention provides animals, cells or cell lines having functionally disrupted endogenous Tpl2, methods, and compositions for the treatment of inflammatory diseases that are induced by various biological and pharmacological agents.
In one aspect of the invention, a knock out animal having a functionally disrupted endogenous Tpl2 gene is provided. The knock out animal can be created by the insertion of an exogenous sequence into said gene or by replacement of part of the endogenous Tpl2 gene with an exogenous sequence which results in the animal that lacks the functional Tpl2 protein. Such an animal can be characterized by, for example, its increased resistance to LPS induced endotoxin shock relative to a wild type animal. The knock out animal can also be characterized by its resistance to TNFxcex1-mediated inflammatory diseases by comparison with an animal that expresses a functional Tpl2 gene. The knock out animal can be homozygous for the functionally disrupted endogenous Tpl2 gene or heterozygous for the functionally disrupted endogenous Tpl2 gene. The knock out animals can be mice, rats, rabbits, goats, pigs or monkeys. Spleen cells or microphages isolated from these animals are also provided. A cell line established from immortalized or transformed macro phages isolated from these animals are also provided.
An animal with a structurally intact Tpl2 gene but functionally disrupted endogenous Tpl2 is also provided. For example, functionally disrupted endogenous Tpl2 can be due to the antisense inhibition of mRNA expression or due to the expression of dominant negative mutant Tpl2. These animals can also be characterized by their resistance to LPS induced endotoxin shock or TNFxcex1-mediated inflanmmatory diseases by comparison with an animal that expresses a functional Tpl2.
In another aspect of the invention, a method of identifying Tpl2 specific inhibitors of endotoxin shock is provided. This method includes (a) administering to a first animal expressing a functional Tpl2 gene, and to a second animal having a functionally disrupted endogenous Tpl2 gene, an amount of an endotoxin shock inducing agent wherein the first animal is susceptible to endotoxin shock induced by the agent; (b) administering to the first and second animals, an amount of a putative Tpl2 inhibitor; and (c) determining whether the first animal shows induced resistance to endotoxin shock comparable to that observed in the second animal. The induced resistance to endotoxin shock in the first animal is due to the Tpl2 specific inhibitor.
In still another aspect of the invention, a method of identifying an antagonist to inflammation that is dependent upon a functional Tpl2 gene, is provided. This method includes the following steps: (a) administering to a first animal that expresses a functional Tpl2 gene and to a second animal having a functionally disrupted endogenous Tpl2 gene, an effective amount of an agonist that induces a greater inflammatory response in the first animal than in the second animal; (b) administering to the first and second animals of step (a) a putative Tpl2 inhibitor; and (c) determining the level of inflammatory response in the first animal compared to that of the second animal after step (b). The agonist can be a biological agent or it can be a pharmacological agent such as bleomycin.
In yet another aspect of the invention, a method of determining whether a compound that is known to inhibit Tpl2 activity in vitro also inhibits LPS induced endotoxin shock by specifically inhibiting Tpl2 in vivo. The method includes: (a) administering to a first animal expressing a functional Tpl2 gene and to a second animal having functionally disrupted endogenous Tpl2 gene an effective amount of LPS to induce endotoxin shock in the first animal; (b) administering to the first and second animals an effective amount of the compound that is known to inhibit Tpl2 activity in vitro; and (c) determining whether the first animal shows reduced endotoxin shock comparable to that in the second animal.
In yet another aspect of the invention, a method of determining whether a compound that is known to inhibit Tpl-2 activity in vitro also inhibits TNFxcex1-mediated inflammatory diseases is provided. This method includes: (a) administering to a first animal expressing a functional Tpl2 gene and to a second animal having functionally disrupted endogenous Tpl2 gene an effective amount an agent that induces TNFxcex1 production or TNFxcex1-mediated inflammatory response; (b) administering to the first and second animals an effective amount of the compound that is known to inhibit Tpl2 activity in vitro; and (c) determining whether the first animal shows reduced inflammatory response comparable to that in the second animal. Inflammatory diseases include Rheumatoid Arthritis.
In a further aspect, a method of treating or preventing TNFxcex1-mediated inflammatory diseases or LPS induced endotoxin shock in an animal is provided which includes: (a) transfecting bone marrow derived cells in vitro with a DNA construct which encodes sequences that interferes with the expression or function of the endogenous Tpl2 in said cells; and (b) administering an effective amount of said cells after step (a) to the animal. The sequences can be Tpl2 kinase mutant sequences or Tpl2 antisense sequences.
In another aspect of the invention a composition having a DNA construct which encodes sequences that interfere with the expression or function of the endogenous Tpl2 is provided. The sequences include Tpl2 sequences carrying one or more dominant negative mutations and antisense Tpl2 sequences.
In further aspects of the invention, nucleic acids and polypeptides which have been isolated (i.e., altered xe2x80x9cby the hand of manxe2x80x9d from its natural state, or if it occurs in nature, it has been changed or removed from its original environment) are provided. In one aspect, an isolated Tpl2 polypeptide is provided which can be: (a) an isolated polypeptide comprising an amino acid sequences having at least 95% identity to the amino acid sequence of SEQ ID NO: 2 or 4 over the entire length of SEQ ID NO: 2 or 4; (b) an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or 4; (c) an isolated polypeptide that is the amino acid sequence of SEQ ID NO: 2 or 4; or (d) a polypeptide that is encoded by a recombinant polynucleotide comprising the polyncleotide sequence of SEQ ID NO: 1 or 3.
In another aspect of the invention, an isolated Tpl2 polynucleotide is provided which can be: (a) an isolated polynucleotide comprising a polynucleotide sequence encoding a polypeptide that has at least 95% identity to the amino acid sequence of SEQ ID NO: 2 or 4, over the entire length of SEQ ID NO: 2 or 4; (b) an isolated polynucleotide comprising a polynucleotide sequence that has at least 95% identity over its entire length to a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 or 4; (c) an isolated polynucleotide comprising a nucleotide sequence that has at least 95% identity to that of SEQ ID NO: 1 or 3 over the entire length of SEQ ID NO: 1 or 3; (d) an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 or 4; (e) an isolated polynucleotide that is the polynucleotide of SEQ ID NO: 1 or 3; (f) an isolated polynucleotide of at least 30 nucleotides in length obtainable by screening an appropriate library under stringent hybridization conditions with a probe having the sequence of SEQ ID NO: 1 or 3 or a fragment of SEQ ID NO: 1 or 3 of at least 30 nucleotides in length; or (g) a polynucleotide sequence complementary to said isolated polynucleotide of (a), (b), (c), (d), (e) or (f).